The overall goal of this research project is to develop on-chip, multiplexed templated biosynthesis methods for the fabrication of (i) RNA aptamer microarrays and (ii) unnatural protein microarrays (uPMs) for use in surface plasmon resonance imaging (SPRI) bioaffinity sensing. SPRI has become a mainstay tool for multiplexed detection of DNA, RNA, and protein biomarkers, and recent advances in SPRI such as surface plasmon resonance phase imaging (SPR-PI) and nanoparticle-enhanced SPR-PI have increased the sensitivity of these measurements for in vitro biomarker detection at extremely low (picomolar to femtomolar) concentrations in microliter sample volumes. Most multiplexed SPRI protein biosensing measurements employ either antibody or DNA aptamer microarrays; the use of RNA aptamer microarrays has been limited due to the significant effort required for the off-chip synthesis and purification of multiple RNA sequences. In this research effort, on-chip methods for the fabrication of self-assembled RNA aptamer microarrays with multiplexed RNA polymerase surface transcription reactions in a small volume microfluidic format will be developed for use with SPR-PI to detect protein biomarkers at picomolar concentrations; RNA split-aptamer assays will be used with nanoparticle-enhanced SPR-PI to detect protein biomarkers at sub-picomolar concentrations and also to detect small molecule metabolites such as ATP. Additionally, we will develop novel on-chip methods for the templated biosynthesis of uPMs - protein microarrays where each of the immobilized proteins contains one or more unnatural amino acids (uAAs). The incorporation of uAAs into proteins can be used to enhance the stability of protein-protein interactions and to incorporate additional reactive moieties, such as alkyne-containing uAAs for surface click attachment chemistry or benzophenone-containing uAAs for the photocapture of bioaffinity targets. The uPMs will be fabricated from DNA templates in an on-chip multiplexed biosynthesis that will employ a combination of ribozyme charging of tRNA with uAAs and a modified in vitro transcription and translation (IVTT) protocol. These uPMs will be coupled to SPRI to measure the enhanced bioaffinity adsorption of target proteins. The first demonstration projects will use both SPRI and fluorescence (where appropriate) to study the fabrication of modified EGFP/CFP microarrays, the incorporation of click attachment chemistry for IVTT protein microarrays, and the photocapture of a bioaffinity target protein with a probe protein by incorporating a benzophenone-modified UAA near the specific protein- protein interaction region. These two simple yet powerful on-chip synthesis methods for creating of RNA aptamer and unnatural protein microarrays will enhance the specificity and selectivity of our SPRI bioaffinity sensing measurements.